This is a competitive renewal of DK38712-20 entitled Regulating IRS1 by Ser/Thr Phosphorylation. The renewal focuses upon the regulation of IRS1 signaling cascades in mouse-based experimental systems. The decreased physiologic response to insulin (insulin resistance) is key to the initiation and progression of type 2 diabetes (T2DM). Post-receptor responsiveness to insulin in muscle, liver and other tissues, including the CNS, depends upon the insulin receptor substrate proteins, IRS1 and IRS2. Tyrosine phosphorylation of IRS-proteins by the insulin receptor recruits the phosphatidylinositol 3' kinase (PI 3-kinase) to a fraction of the cellular IRS1/IRS2 pool, activating downstream kinases that mediate the cellular insulin response. Although the interactions of numerous genetic and environmental factors promote T2DM, negative regulation of IRS1 and IRS2 signaling by serine or threonine phosphorylation has emerged as a common mechanism of insulin resistance. However, the experimental verification and exploration of this mechanism in whole animals has been limited by the large number of serine (Ser) and threonine (Thr) phosphorylation sites found in IRS1 and IRS2. Given this biological complexity, insulin resistance in many studies has been attributed to the phosphorylation of one or a few Ser/Thr residues in IRS1 to which commercially-produced antibodies are available. However, a positive contribution to the normal insulin response of phosphorylation at particular Ser/Thr sites cannot be ruled out form current data. Numerous cell-based and animal or human studies have shown that Ser307 in rodent Irs1 (human Ser312) is phosphorylated during insulin resistance. Phosphorylation of nearby Ser302 in rodent Irs1 is similarly implicated in insulin resistance, especially that stemming from nutrient excess and activation of signaling by the mTORC1 holo-kinase (mammalian target of rapamycin complex 1). Our laboratory has developed the experimental systems to directly investigate the effect of these and other modifications upon the insulin sensitivity of mice. The mechanism(s) by which Ser307 and Ser302 in rodent Irs1-or other phosphorylation sites-regulate insulin signaling will be investigated in this proposal under physiologic conditions of health or obesity that progresses to insulin resistance and diabetes using new mouse genetic models and a set of 25 phospho- and site-specific monoclonal antibodies, as outlined in the following Specific Aims: 1. Define the Ser/Thr-phosphorylation profile of IRS1 associated with PI 3-kinase signaling. 2. Establish phospho-mimetic E307Irs1 and E302Irs1 knock-in mice to model the effect of phosphorylation at these positions of Irs1. 3. Identify the mTORC1-dependent phosphorylation sites in IRS1 that regulate insulin signaling and glucose tolerance in mice.